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Target specificity of the E3 ligase LUBAC for ubiquitin and NEMO relies on different minimal requirements.

Smit JJ, van Dijk WJ, El Atmioui D, Merkx R, Ovaa H, Sixma TK - J. Biol. Chem. (2013)

Bottom Line: The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway.Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin.Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry and.

ABSTRACT
The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.

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HOIPRBR-LDD mediated ubiquitin chain formation with N-terminal modified synthetic ubiquitins.A, E3 ligase constructs used in this study. HOIP ubiquitin-like domain (UBL), Npl4 zinc finger (ZF), ubiquitin-associated domain (UBA), LDD, and a RBR consisting of two RING domains (R1 and R2) and an in-between RING domain (IBR). The domain borders of the ubiquitin-like domain, ZF, UBA, and RBR domains are drawn to scale according to Uniprot definitions. B, schematic representation of the N-terminal residues of the synthetic N-terminal-modified ubiquitins. Construct names represent the wild type amino acids between parentheses and additional amino acids by their three-letter code. Norleucine (Nle) was used as a steric equivalent of Met-1, and 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx) were designed to position an amino group at a similar position compared with the wild type N terminus. Ubiquitin mutants that were not used by HOIP have a gray background. C, HOIPRBR-LDD (E3) functions together with Ube2L3 (E2) to mediate free ubiquitin chain formation with ubiquitin Met-1 point mutants after 30 min. D, acceptor assay with TAMRAubiquitin loaded on the E2 Ube2L3. HOIPRBR-LDD does not transfer TAMRAubiquitin from the E2 onto the N-terminally shortened or elongated ubiquitins.
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Figure 1: HOIPRBR-LDD mediated ubiquitin chain formation with N-terminal modified synthetic ubiquitins.A, E3 ligase constructs used in this study. HOIP ubiquitin-like domain (UBL), Npl4 zinc finger (ZF), ubiquitin-associated domain (UBA), LDD, and a RBR consisting of two RING domains (R1 and R2) and an in-between RING domain (IBR). The domain borders of the ubiquitin-like domain, ZF, UBA, and RBR domains are drawn to scale according to Uniprot definitions. B, schematic representation of the N-terminal residues of the synthetic N-terminal-modified ubiquitins. Construct names represent the wild type amino acids between parentheses and additional amino acids by their three-letter code. Norleucine (Nle) was used as a steric equivalent of Met-1, and 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx) were designed to position an amino group at a similar position compared with the wild type N terminus. Ubiquitin mutants that were not used by HOIP have a gray background. C, HOIPRBR-LDD (E3) functions together with Ube2L3 (E2) to mediate free ubiquitin chain formation with ubiquitin Met-1 point mutants after 30 min. D, acceptor assay with TAMRAubiquitin loaded on the E2 Ube2L3. HOIPRBR-LDD does not transfer TAMRAubiquitin from the E2 onto the N-terminally shortened or elongated ubiquitins.

Mentions: The linear ubiquitin chain-forming activity of HOIP is autoinhibited by its N terminus (28, 29). To release the inhibited state, full-length HOIP needs to form a complex via its UBA domain with the UBL domain of either HOIL-1L or Sharpin (32). The different HOIP-containing complexes that consist of either HOIP·HOIL-1L or HOIP·Sharpin or HOIP·HOIL-1L/Sharpin can all activate the NF-κB pathway (4, 9, 30, 31). However, the isolated HOIP RBR-LDD domain, which lacks the HOIP N terminus, is sufficient for the formation of free linear ubiquitin chains in vitro in the absence of the other LUBAC components (Fig. 1A) (28, 29).


Target specificity of the E3 ligase LUBAC for ubiquitin and NEMO relies on different minimal requirements.

Smit JJ, van Dijk WJ, El Atmioui D, Merkx R, Ovaa H, Sixma TK - J. Biol. Chem. (2013)

HOIPRBR-LDD mediated ubiquitin chain formation with N-terminal modified synthetic ubiquitins.A, E3 ligase constructs used in this study. HOIP ubiquitin-like domain (UBL), Npl4 zinc finger (ZF), ubiquitin-associated domain (UBA), LDD, and a RBR consisting of two RING domains (R1 and R2) and an in-between RING domain (IBR). The domain borders of the ubiquitin-like domain, ZF, UBA, and RBR domains are drawn to scale according to Uniprot definitions. B, schematic representation of the N-terminal residues of the synthetic N-terminal-modified ubiquitins. Construct names represent the wild type amino acids between parentheses and additional amino acids by their three-letter code. Norleucine (Nle) was used as a steric equivalent of Met-1, and 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx) were designed to position an amino group at a similar position compared with the wild type N terminus. Ubiquitin mutants that were not used by HOIP have a gray background. C, HOIPRBR-LDD (E3) functions together with Ube2L3 (E2) to mediate free ubiquitin chain formation with ubiquitin Met-1 point mutants after 30 min. D, acceptor assay with TAMRAubiquitin loaded on the E2 Ube2L3. HOIPRBR-LDD does not transfer TAMRAubiquitin from the E2 onto the N-terminally shortened or elongated ubiquitins.
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Related In: Results  -  Collection

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Figure 1: HOIPRBR-LDD mediated ubiquitin chain formation with N-terminal modified synthetic ubiquitins.A, E3 ligase constructs used in this study. HOIP ubiquitin-like domain (UBL), Npl4 zinc finger (ZF), ubiquitin-associated domain (UBA), LDD, and a RBR consisting of two RING domains (R1 and R2) and an in-between RING domain (IBR). The domain borders of the ubiquitin-like domain, ZF, UBA, and RBR domains are drawn to scale according to Uniprot definitions. B, schematic representation of the N-terminal residues of the synthetic N-terminal-modified ubiquitins. Construct names represent the wild type amino acids between parentheses and additional amino acids by their three-letter code. Norleucine (Nle) was used as a steric equivalent of Met-1, and 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx) were designed to position an amino group at a similar position compared with the wild type N terminus. Ubiquitin mutants that were not used by HOIP have a gray background. C, HOIPRBR-LDD (E3) functions together with Ube2L3 (E2) to mediate free ubiquitin chain formation with ubiquitin Met-1 point mutants after 30 min. D, acceptor assay with TAMRAubiquitin loaded on the E2 Ube2L3. HOIPRBR-LDD does not transfer TAMRAubiquitin from the E2 onto the N-terminally shortened or elongated ubiquitins.
Mentions: The linear ubiquitin chain-forming activity of HOIP is autoinhibited by its N terminus (28, 29). To release the inhibited state, full-length HOIP needs to form a complex via its UBA domain with the UBL domain of either HOIL-1L or Sharpin (32). The different HOIP-containing complexes that consist of either HOIP·HOIL-1L or HOIP·Sharpin or HOIP·HOIL-1L/Sharpin can all activate the NF-κB pathway (4, 9, 30, 31). However, the isolated HOIP RBR-LDD domain, which lacks the HOIP N terminus, is sufficient for the formation of free linear ubiquitin chains in vitro in the absence of the other LUBAC components (Fig. 1A) (28, 29).

Bottom Line: The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway.Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin.Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry and.

ABSTRACT
The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.

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